Project Summary The kidney plays a key role in maintaining plasma [K+], with distal segments of the nephron fine-tuning K+ secretion to keep it in the normal range. We previously proposed that the renal distal convoluted tubule (DCT) plays a key role by sensing plasma [K+]. Decreasing plasma [K+] by dietary K+ restriction activates the WNK- SPAK/OSR1-NCC pathway, and increased NaCl reabsorption though NCC reduces delivery of sodium to, and possibly remodels, distal K+ secreting segments to lower K+ secretion. The disease Familial Hyperkalemic Hypertension (FHHt) is caused by increased NCC activation due to mutations in WNKs, Cullin 3 (CUL3), and KLHL3. The Cullin Ring Ligase (CRL) complex, composed of the scaffold CUL3, the substrate adaptor KLHL3, and the ligase RING, degrades WNKs. The effects of mutant CUL3, produced by skipping of exon 9 which causes internal deletion of 57 amino acids (CUL3-?9), are controversial. CUL3-?9 triggers its own degradation in vitro, and also in a mouse model of CUL3 FHHt. Thus, the prevailing model is that CUL3-?9 causes FHHt by inducing CUL3 haploinsufficiency. Our preliminary data in CUL3 heterozygote mice and a new mouse model of CUL3-?9 FHHt do not support this, and we hypothesize that CUL3-?9 exerts dominant effects to cause FHHt and dysregulate the plasma [K+] sensor. We propose that CUL3-?9 causes FHHt by a combined effect of lowering abundance of itself and of KLHL3. Our data suggest that NKCC2 activation along the thick ascending limb (TAL) may also contribute to FHHt. Finally, we previously generated kidney-specific CUL3 knockout (KO) mice, and found that they display a severe phenotype (polyuria and chronic kidney disease), with defects along multiple nephron segments. Our overall aim is to determine the mechanisms underlying CUL3-?9-mediated FHHt, and gain insight into CUL3 function in the kidney. In Aim 1 we will determine the effects of CUL3-?9 expression and CUL3 KO specifically along DCT to determine whether CRL disruption along DCT is sufficient to cause FHHt. We will determine the effects of CRL disruption on KLHL3 in mice, since we found CUL3-?9 inappropriately degrades it in cultured cells. We will also directly test whether mice with lower abundance of CUL3 and KLHL3 develop FHHt. In Aim 2 we will determine whether remodeling of K+-secreting segments occurs in FHHt mediated by CUL3-?9, and examine the effects of CRL disruption on NKCC2 activity. Some models suggest that CUL3-?9 leads to dramatically lower CRL activity, but data suggest this would be lethal. We propose that CUL3-?9 may exert unique effects that cause it to preferentially degrade certain CRL adaptors. Therefore, in Aim 3 we will examine effects of CUL3-?9 on other CRL adaptors and substrates in our mouse models and in primary cell culture.